Ryl. Tsai et Rr. Reed, IDENTIFICATION OF DNA RECOGNITION SEQUENCES AND PROTEIN-INTERACTION DOMAINS OF THE MULTIPLE-ZN-FINGER PROTEIN ROAZ, Molecular and cellular biology (Print), 18(11), 1998, pp. 6447-6456
Roaz, a rat C2H2 zinc finger protein, plays a role in the regulation o
f olfactory neuronal differentiation through its interaction with the
Olf-1/EBF transcription factor family. An additional role for the Roaz
/Olf-1/ EBF heterodimeric protein is suggested by its ability to regul
ate gene activation at a distinct promoter lacking Olf-1/EBF-binding s
ites. Using an in vitro binding-site selection assay (Selex), we demon
strate that Roaz protein binds to novel inverted perfect or imperfect
repeats of GCACCC separated by 2 bp. We show that Roaz is capable of b
inding to a canonical consensus recognition sequence with high affinit
y (K-d = 3 nM). Analysis of the structural requirement for protein dim
erization and DNA binding by Roaz reveals the role of specific zinc fi
nger motifs in the Roaz protein for homodimerization and heterodimeriz
ation with the Olf-1/EBF transcription factor, The DNA-binding domain
of Roaz is mapped to the N-terminal 277 amino acids, containing the fi
rst seven zinc finger motifs, which confers weak monomeric binding to
a single half site and a stronger dimeric binding to the inverted repe
at in a binding-site-dependent manner. Full-length protein can form di
mers on both the inverted repeat and direct repeat but not on a single
half site. These findings support the role of the TFIIIA-type Zn fing
ers in both protein-protein interaction and protein-DNA interaction an
d suggest distinct functions for specific motifs in proteins with a la
rge number of zinc finger structures.